The cyclic nucleotides, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), were discovered decades ago and represent one of the most important second messenger pathway within cells. It is well established that the regulation of intra-cellular cGMP pools have substantial impact on physiology, and pathophysiology and is one basic principle of pharmacological intervention (Eugenov et al. 2006; Schmidt et al. 2009). Nitrates and PDE5 inhibitors (PDE5i) which could increase intra-cellular cGMP levels are therefore already approved therapies for Angina, and Pulmonary Arterial Hypertension (PAH) or Erectile Dysfunction (ED), respectively. More recently discovered sGC stimulators and activators, are in advanced states of clinical development for PAH and Heart Failure. Therefore, targeting the NO/cGMP pathway by either cGMP production (nitrates, sGC stimulators, sGC activators) or cGMP break-down (PDE5i) became a very effective pharmacological intervention strategy in various diseases.
On a molecular level, NO-production results in stimulation of the soluble guanylate cyclase (sGC) resulting in enhanced cGMP formation. Consecutively, cGMP regulates different downstream targets, mainly cGMP regulated protein kinases (G-Kinases), cGMP-regulated phosphodiesterases (PDEs) and cGMP regulated ion channels which translates the NO-signal and rise in cGMP and in a decrease of intracellular free calcium. Therefore, the most prominent response of increasing intracellular cGMP, especially in the Smooth Muscle Cell (SMC), is relaxation. In addition, antiproliferative, antifibrotic or proapoptotic effects of cGMP are discussed and might expand the treatment options for PDE5 inhibitors (Sandner et al. 2007, Schmidt et al. 2009). More recently some lines of evidence showed that PDE5 inhibitors could also influence chloride secretion via the chloride channel CFTR and might be useful for the treatment of Cystic Fibrosis (Clarke 2008).
Cystic fibrosis (CF) is one of the most prevalent genetic disorders, caused by mutation of a single gene, the CFTR-channel, affecting 1 out of 2500-3000 newborns. In this disease, abnormal ion transport across the respiratory epithelia leads to dehydrated airway surface and viscous and poorly-cleared mucus. This contributes to chronic infections of the airways and high morbidity and early mortality. Up to now, the treatment is mainly focused on anti-infective treatment and lung transplantation but no causal therapy focusing on the correction and potentiation of impaired CFTR function is available.
On the molecular level a mutation in the CFTR gene results in CF. A broad variety of CF-causing mutations in the CFTR gene have been identified. However, the most prevalent mutation is a deletion of the phenylalanine in position 508 of the CFTR amino acid sequence, and is termed as ΔF508-CFTR. This mutation occurs in approximately 70%-80% of the cases of CF and is associated with a severe disease.
The deletion of residue 508 in ΔF508-CFTR prevents the mature protein from correct processing and folding. This missfolded CFTR could not, or not completely, exit the ER, and traffic to the plasma membrane. As a result, the number of channels present in the membrane in CFTR-patients is far less than observed in cells expressing wild-type CFTR. In addition the mutated channel exhibited reduced channel activity. Both, the reduced number of channels in the epithelial membranes and the reduced channel acitivity results in significantly impaired anion transport across epithelia leading to defective ion and fluid transport. This causes an imbalance in lung epithelial fluid transport and finally an excessive accumulation of viscosous mucus in the lungs. Moreover impaiered CFTR-function also influences e.g. pancreatic function, gastro-intestinal functions, liver function, functions of secretory glands or insulin secretion.
In summary, impaired CFTR function by several mutations cause cystic fibrosis. Therefore correction and/or potentiation of CFTR function of these mutations could present a causal treatment option for Cystic Fibrosis (CF).
In addition correction and/or potentiation of CFTR function could present a causal treatment option for pancreatic dysfunction, liver dysfunction, dry mouth, dry eye, Sojegren's syndrome, and CF-induced diabetes.
It was shown, that in lung epithelial cells sildenafil—a potent and selective PDE5i—increased CFTR-driven chloride secretion (Cobb et. al 2003). In addition trafficking and functional activity of mutated CFTR-channels to the cell membrane could be influenced by PDE5i in vitro (Dormer et. al 2005, Carlile et al. 2007, Robert et al. 2008). In line with these findings it was demonstrated that PDE5 inhibitors when used in animal models of Cystic fibrosis (CF) are able to reduce mucin secretin (Mc Pherson 1999) and could influence chloride secretion (Lubamba et al. 2008). Therefore it was hypothesized that PDE5 inhibitors could be used for the treatment of Cystic Fibrosis (Cobb 1999, Lubamba et. al. 2008, Clarke 2008).
However, the use of PDE5 inhibitors is limited since they could only inhibit cGMP degradation. In cases in which NO-dependent cGMP production is low, their efficacy is at least partially impaired. Very interestingly, compounds have been described recently that could overcome this limitation of PDE5 inhibitors via direct stimulation or activation of the sGC. Two classes of compounds have been identified that activate the sGC NO-independently, the heme-dependent sGC stimulators, such as BAY 41-2272 according to compound of the formula (4a), BAY 41-8543 according to compound of the formula (1), BAY 59-3394 according to compound of the formula (2), the compound according to formula (3a), BAY 63-2521 according to compound of the formula (3), and BAY 60-4552 according to compound of the formula (4), and heme-independent sGC activators, such as BAY 58-2667 according to compound of the formula (5), HMR-1766 according to compound of the formula (6), (Stasch et al. 2001, for review see Evgenov et al. 2006), and the sGC activators disclosed in WO 2012/139888, herewith incorporated by reference.
In addition, it has been shown very recently that correction of deltaF508 function, using a corrector compound (VX-809) is not sufficient for a clinically meaningful treatment effect in deltaF508 CF patients (M. Boyle, NACCF-presentation November 2012). However, when VX-809 was combined with a potentiator compound, i.e. VX-770, restoration of deltaF508 function was significantly improved, resulting in a clinical benefit for the CF-patient.
sGC stimulators, sGC activators and/or PDE5 inhbitors in combination with other pharmacological compounds which lead to increased cGMP mobilization, i.e. Nitrates, NO-Donors, Natriumnitroprussid, Nitroglycerine, Isosorbidmononitrate, Isosorbiddinitrate, Molsidomin or SIN-1, inhaled Nitric Oxide (NO), could result in superior clinical benefits for CF patients.
In addition, sGC stimulators, sGC activators and/or PDE5 inhbitors in combination with other molecules, correcting and potentiating i.e. deltaF508 CFTR function, i.e. VX-809, VX-770, VX-661 could significantly enhance the effect of sGC stimulators, sGC activators and PDE5 inhbitors, resulting in superior clinical benefits for CF patients.
In addition, combination of of sGC stimulators, sGC activators and/or PDE5 inhbitors with the current standard of care in CF, i.e. systemic or nebulized antibiotics, Dornase Alpha (rhDNase), hypertonic saline, asthma treatments, could result in superior clinical benefits for CF patients. In addition, combination of of sGC stimulators, sGC activators and/or PDE5 inhbitors with anti-inflammatory drugs, i.e. systemic or nebulized glucocorticoids, serine protease inhibitors, elastase inhibitors, could result in superior clinical benefits for CF patients.
We therefore investigated sGC stimulators and sGC activators, i.e. BAY 41-2272, BAY 60-4552 according to compound of the formula (4a, 4), alone or in combinations with PDE5 inhibitors, i.e. vardenafil, in vivo, in CFTR-transgenic animal models, i.e. in deltaF508 CFTR mice. We compared these effects with correctors (i.e.) VX-809 and potentiators (VX-770). In addition, effective concentrations and dosages of the aforementioned compounds were combined and efficacy in murine CF-models was investigated.
In particular, PDE5 inhibitors, sGC stimulators, sGC activators, potentiators (i.e. VX-770), correctors (i.e. VX-809) alone or combinations thereof, were tested:                in transgenic mice expressing the delta F508CFTR channel, on nasal potential difference, on salivation secretion as on salivation chloride content as descriebed recently (Droebner and Sandner; 2013).        in transgenic mice not expressing the CFTR, on nasal potential difference, on salivation secretion as on salivation chloride content. as descriebed recently (Droebner and Sandner; 2013).        
We could demonstrate that stimulators and activators of the soluble guanylate cyclase, corrected and potentiated CFTR function, presenting a new causal treatment option for CF-patients. In addition, we found that combinations of sGC stimulators or sGC activators with PDE5i showed more than additive effects on correction and potentiation. Effects of sGC stimulators, sGC activators and/or PDE5 inhibitors alone or in combination were superior to the effects seen by correctors, i.e. VX-809. Combination of sGC stimulators or sGC activators and PDE5 inhibitors with correctors, i.e. VX-809 showed completely unexpected overadditive effects.
In summary we discovered that combinations of sGC stimulators or sGC activators and/or PDE5 inhibitors when combined with other treatment options could correct and potentiale CFTR function in vivo in an overadditive mode. Due to this increased efficacy combinations of sGC stimulators, sGC activators and/or PDE5 inhibitors, with correctors having a different mode of action could become a highly effective treatment of Cystic Fibrosis (CF).